日本血吸虫四跨膜蛋白第二亲水基团(TSP2HD)基因合成、表达与免疫原性研究

Gene Synthesis, Expression and Immunogenicity Analysis of TSP_2 Hydrophilic Domain(TSP_2HD) of Schistosoma japonicum

目的合成和表达日本血吸虫四跨膜蛋白第二亲水基团(TSP2HD)基因,并研究其免疫原性。方法采用重叠PCR人工合成日本血吸虫TSP2HD(aa107～aa182)完整基因片段,经测序正确后,将此片段插入表达载体pGEX-4T-3,构建重组表达质粒TSP2HD-PG,转化大肠埃希菌BL21(DE3),获得含重组表达质粒的转化子,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)观察TSP2HD融合蛋白表达情况。用谷胱甘肽(GST)融合蛋白纯化胶(glutathione sepharose 4B)从表达产物裂解上清中纯化GST-TSP2HD融合蛋白,用凝血酶切割融合蛋白,制备纯化的重组TSP2HD蛋白。通过蛋白质印迹(Western blotting)分析重组TSP2HD蛋白与血吸虫病患者血清及血吸虫重感染兔血清的免疫反应性;重组TSP2HD蛋白刺激血吸虫感染小鼠淋巴细胞进行淋巴细胞增殖试验,通过比较实验组与对照组脉冲指数(cpm)值的差异研究重组TSP2HD蛋白的免疫原性。结果经过3轮重叠PCR扩增,获得长228bp的TSP2HD基因,序列分析证实与天然基因序列完全一致。含重组质粒TSP2HD-PG的转化子细菌,经IPTG诱导后表达相对分子质量(Mr)约为34000的可溶性GST-TSP2HD融合蛋白。凝血酶切割GST-TSP2HD融合蛋白获得纯化的重组TSP2HD蛋白。Western blotting分析证明,重组表达蛋白可被血吸虫重感染兔血清和血吸虫病患者血清识别,有较好的免疫反应性;重组TSP2HD蛋白能刺激血吸虫感染小鼠脾细胞增殖,实验组cpm值明显高于对照组,两者间差异有统计学意义(P<0.01)。结论日本血吸虫TSP2HD基因合成及表达获得成功,重组TSP2HD蛋白有天然免疫原性。

Objective To synthesize and express the gene of TSP2 hydrophilic domain of Schistosoma japonicum, and investigate the immunogenicity of the recombinant TSP2HD protein. Methods The whole DNA fragment encoding the TSP2 hydrophilic domain was synthesized by overlapping PCR, and confirmed by DNA sequencing. The recombinant plasmid TSP2HD-PG was constructed by inserting the purified TSP2HD DNA fragment into expression vector pGEX-4T-3 and the GST-TSP2HD fusion protein was expressed by transforming the recombinant plasmid TSP2HD-PG into Escherichia coli BL21 （DE3） and induced the recombinant with isopropyl β-D-1-thiogalactopyranoside （IPTG） . The expressing situation of fusion protein was analyzed by SDS-PAGE. The GST-TSP2HD fusion protein was purified by affinity chromatography with glutathione sepharose 4B gel, and the purified recombinant TSP2HD protein was prepared by digesting the GST-TSP2HD fusion protein with thrombin. The immuno-response of the recombinant TSP2HD recognized by the pool sera of schistosomiasis patients and the pool sera of heavily infected rabbits was explored by Western blotting analysis. The immunogenicity of the recombinant TSP2HD was investigated by comparing the difference of counts per minute （cpm） value of lymphocyte proliferation test between experiment group and control group. Results A 228 bp of TSP2HD gene fragment was obtained after overlapping PCR of three times and its DNA sequence was confirmed by DNA sequencing, which was same to one of the native TSP2HD. The recombinant containing recombinant plasmid TSP2HD-PG expressed a soluble fusion protein of GST-TSP2HD （Mr≈34 000） after being induced with IPTG. The purified recombinant TSP2HD protein was obtained through digesting the GST-TSP2HD fusion protein with thrombin. The recombinant TSP2HD was recognized by pool sera of schistosomiasis patients and pool sera of infected rabbits, indica ting that the recombinant TSP2HD has a good response activity. The recombinant TSP2HD also stimulated proliferation of lymphocytes in infected mouse, the cpm value of experiment group was higher than that of the control （P〈0.01）. Conclusion The Sj TSP2HD gone has been synthesized and expressed with immunogenicity which is similar to that of the native antigen.